Efficacy of pamidronate in reducing skeletal events in patients with advanced multiple myeloma. Myeloma Aredia Study Group.

BACKGROUND: Skeletal complications are a major clinical manifestation of multiple myeloma. These complications are caused by soluble factors that stimulate osteoclasts to resorb bone. Bisphosphonates such as pamidronate inhibit osteoclastic activity and reduce bone resorption. METHODS: Patients with stage III multiple myeloma and at least one lytic lesion received either placebo or pamidronate (90 mg) as a four-hour intravenous infusion given every four weeks for nine cycles in addition to antimyeloma therapy. The patients were stratified according to whether they were receiving first-line (stratum 1) or second-line (stratum 2) antimyeloma chemotherapy at entry into the study. Skeletal events (pathologic fracture, irradiation of or surgery on bone, and spinal cord compression), hypercalcemia (symptoms or a serum calcium concentration > or = 12 mg per deciliter [3.0 mmol per liter]), bone pain, analgesic-drug use, performance status, and quality of life were assessed monthly. RESULTS: Among 392 treated patients, the efficacy of treatment could be evaluated in 196 who received pamidronate and 181 who received placebo. The proportion of patients who had any skeletal events was significantly lower in the pamidronate group (24 percent) than in the placebo group (41 percent, P < 0.001), and the reduction was evident in both stratum 1 (P = 0.04) and stratum 2 (P = 0.004). The patients who received pamidronate had significant decreases in bone pain and no deterioration in performance status and quality of life. Pamidronate was tolerated well. CONCLUSIONS: Monthly infusions of pamidronate provide significant protection against skeletal complications and improve the quality of life of patients with stage III multiple myeloma.

Elimination of T cells from human peripheral blood and bone marrow using a cocktail of three anti-T cell immunotoxins.

Four anti-T cell monoclonal antibodies were coupled to ricin-A and tested for their ability to kill T cells in peripheral blood and bone marrow using a clonogenic assay to quantify T cell survival. The immunotoxins (IT) prepared from RFT11 (CD2) and WT1 (CD7) antibodies were the most toxic to peripheral blood T cells. The immunotoxin prepared from RFT1 (CD5) was the next most efficient toxin and the immunotoxin prepared from RFT8 (CD8) was the least toxic. When these reagents were applied to peripheral blood cells at 3 x 10(-8) M the number of T cell colonies was reduced by an average of 95%, 94%, 84% and 50%, respectively. Peripheral blood T cells from different donors showed marked variability in their sensitivity to ITs. However, a cocktail of three ITs prepared from RFT11, WT1 and RFT1 gave superior and consistent killing (mean 99.9%; range 99.8-100%) of peripheral blood T cells from six donors. When this cocktail was applied to bone marrow cells from six donors, an average of 99.6% (range 99.5-99.8%) of the T cells were killed. Under the same conditions there was little or no reduction in the number of normal haematopoietic progenitors (CFU-GM, CFU-GEMM, CFU-Meg and BFU-E).

Haemorrhagic cystitis in bone marrow transplantation patients: possible increased risk associated with prior busulphan therapy.

Following bone marrow transplantation employing conditioning including 'high-dose' cyclophosphamide, 65 patients were studied for the subsequent development of symptomatic haemorrhagic cystitis. There was no protection from the urothelial toxicity of cyclophosphamide metabolites afforded by the concurrent administration of 2-mercaptoethane sodium sulphonate (mesna) if timing errors in administration were made. Other factors which might increase the risk of haemorrhagic cystitis due to cyclophosphamide administration include the prior administration of busulphan to patients with chronic granulocytic leukaemia, in whom the incidence of haemorrhagic cystitis was 36% compared with 4% in all other patients. We have also investigated the use of intravesical prostaglandin E2 as a treatment for haemorrhagic cystitis in eight patients, two of whom appeared to obtain major benefit.

JM, a Thy-ALL cell line produces both inhibitory and stimulatory lymphokines for bone marrow progenitor cells.

Normal and malignant T cells as well as T-cell hybridomas have frequently been reported to produce factors which stimulate the growth of committed hemopoietic progenitors. One previous report described a lymphokine produced by a T-cell clone which inhibited hemopoietic progenitor cell proliferation. We now describe the simultaneous production of two activities by a Thy-ALL cell line (JM), a sub-line of Jurkat. Two sets of culture conditions were used: the Fauser & Messner and Iscove's assays. We have been able to separate both inhibitory and stimulatory factors for the growth of multipotent and committed bone marrow progenitors (CFU-GEMM, BFU-E, CFU-E and CFU-GM). The stimulatory factor has an apparent mol. wt of less than 30,000 and the inhibitor an apparent mol. wt of 65-80,000. The growth promoting activity for BFU-E and CFU-GEMM could replace that of phytohemagglutinin stimulated leucocyte conditioned medium (PHA-LCM). We do not know if the production of both activities is due to the malignant phenotype or if there is a normal counterpart to JM that could produce both inhibitory and stimulatory factors.

Graft rejection following HLA matched T-lymphocyte depleted bone marrow transplantation.

Bone marrow graft rejection following HLA-matched bone marrow transplantation (BMT) for leukaemia has been a rare problem. However, with the introduction of T-lymphocyte depleted BMT, graft rejection is recognized as a new complication. At the Royal Free Hospital (RFH) in London T-depletion is achieved using two monoclonal antibodies with complement mediated lysis. The methodology was extended to other centres and in total 56 patients have received T-depleted, HLA matched BMT. Twelve of 56 patients have had graft rejection. At the RFH three of 41 (7%) patients have had rejection whereas at collaborating centres nine of 15 (60%) patients have had rejection. We have investigated these rejections in order to identify factor(s) responsible. Rejection was not restricted by patient or donor characteristics, nor disease status. Patient management, chemotherapy conditioning, efficiency of T-depletion, graft versus host disease (GvHD), and infection post BMT, were not consistently implicated. The major difference between the RFH and all other centres was in the radiotherapy (RT) conditioning: The RFH prescribed a single fraction of 7.5 Gy total body irradiation (TBI) whilst collaborating centres gave 10 or 12 Gy fractionated TBI. We conclude that the different incidence of rejection (7% v. 60%) relates primarily to the RT conditioning although the mechanisms(s) of rejection remain unknown. We conclude that where T-depleted BMT is used, compensation by more intensive RT conditioning is required in order to avert graft rejection.

T lymphocyte regeneration after transplantation of T cell depleted allogeneic bone marrow.

The regeneration of T cell subsets was studied with double immunofluorescence marker methods in 37 patients who received HLA matched T lymphocyte depleted bone marrow transplants (BMT) as part of the treatment for their haematological disease. A cocktail of anti-pan-T (CD6: MBG6) and anti-suppressor/cytotoxic-T cell (CD8: RFT8) monoclonal antibodies was used with rabbit serum as a source of cytolytic complement to achieve selective T cell lysis. The T8+ cells reached low normal values around 60 days post-transplant and remained within the normal range throughout the study (greater than 150 days). This observation is in contrast to our previously published results in patients who, after receiving BMT without efficient T cell depletion, had increased numbers of circulation T8+ cells from 60 days post-transplant. In the present study Leu-7+, RFT8- cells reached normal values rapidly but the reconstitution of T4+ lymphocytes was slow: low normal levels were reached only around day 150 following BMT. The degree of graft-versus-host disease (GVHD) seemed to be related to the number of residual T cells infused: two of the three patients who received the highest numbers of T cells developed Grade II III; otherwise GVHD was minimal. Among the clinical parameters studied cytomegalovirus (CMV) immune status moderately influenced reconstitution: at 55-90 days post-transplant T8+ cells were present at the upper normal levels in seven out of 15 patients receiving BMT from CMV seronegative donors, but in none of the 16 individuals receiving BMT from seropositive donors. CMV related complications were relatively uncommon. Thus the most significant factor in preventing 'T8+ cell overshoot' and T cell imbalance during regeneration appears to be the depletion of T (including T8+) lymphocytes from marrow. The differences of T8+ cell reconstitution in this and previous studies may reflect a different regeneration pattern from T cell precursors as opposed to inoculated mature T cells.

Virological and serological diagnosis of cytomegalovirus infection in bone marrow allograft recipients.

To detect cytomegalovirus (CMV) infections, a total of 1,074 cultures of urine, saliva, or blood were collected weekly from 43 consecutive patients undergoing allogeneic bone marrow transplantation. Twenty-three patients were seronegative before transplant and primary infection occurred in 2 (9%). Twenty patients were initially seropositive and recurrent infections occurred in 5 (25%). Three patients in the recurrent group had proven CMV pneumonitis; viraemia was detected in two recipients, while the third had CMV isolated only from bronchial lavage fluid. The serological response of the 43 patients was defined by testing 559 serial sera for specific IgG and IgM antibodies by radioimmunoassay. Passive acquisition of IgG antibodies from blood products was found in 78% of initially seronegative recipients. One patient with primary infection responded in a pattern typical of immunocompetent individuals with long-term production of specific IgG and transient production of specific IgM antibodies. The second patient also had a typical response, but this was delayed until several weeks after the start of virus excretion. In patients with recurrent infections, specific IgM production did not correlate with episodes of virus excretion. Three of five such patients failed to mount a specific IgM response, and these were the only patients in the study to develop CMV pneumonitis. We conclude that CMV infection in bone marrow recipients can only be diagnosed by detection of virus; therefore, the ability of these patients to mount humoral immune responses should not be relied upon for diagnostic purposes.

Studies of sub-human primate (marmoset) pluripotent hemopoietic stem cells (CFU-GEMM) in vitro.

Pluripotent hemopoietic progenitor cells (CFU-GEMM) grow in vitro from marmoset bone marrow using a modified human CFU-GEMM assay. Characteristics of growth are similar to those reported in the human CFU-GEMM assay. The number of CFU-GEMM/10(5) marrow cells from marmoset bone marrow is approximately four times that grown in human marrow in our laboratory. Data concerning EPO and other requirements for growth of CFU-GEMM demonstrate an assay for the pluripotent hemopoietic progenitor in the marmoset. This assay may be useful in designing preclinical primate bone marrow transplant experiments.

Rapid diagnosis of cytomegalovirus infection in immunocompromised patients by detection of early antigen fluorescent foci.

Cell-cultures of cytomegalovirus (CMV) were fixed after 24 hours' incubation and examined by a monoclonal antibody based immunofluorescence method for the detection of CMV-specific early antigens. 385 urine, saliva, or blood samples from 63 immunocompromised patients were inoculated onto cell-cultures. Comparison with the results of conventional cell-cultures in patients who remained uninfected showed that the new technique had a specificity of 100%. The sensitivity was 80%. This immunofluorescence method gave positive results 27h after inoculation of the specimens instead of the mean of 17 X 5 days with the conventional method based on detection of cytopathic effect. 3 saliva samples, from patients who had previously excreted CMV, reacted in the immunofluorescence method but CMV, reacted in the cell-cultures-perhaps because the assay identified defective, interfering particles in these samples. The monoclonal antibodies were also used successfully in another immunofluorescence system to diagnose cytomegalovirus pneumonitis in 3 patients by testing material obtained by bronchoalveolar lavage.

Aplastic crisis and other effects of the human parvovirus infection.

Human parvovirus infections are common, provoke aplastic crises in patients with congenital haemolytic anaemia and cause fifth disease. An unknown proportion of the infections are subclinical. Parvoviraemia occurs in the early acute stage of infection and specific IgM can be detected during recovery. Most patients commencing an aplastic crisis are viraemic, but fifth disease arises after the viraemia. Serological tests for HPV, available at a small but increasing number of laboratories, will soon be complemented by tests for HPV DNA sequences present in blood, marrow and at other sites. Human parvovirus infection is occasionally fatal in patients with severe forms of congenital haemolytic anaemias and further study may reveal other unusual serious consequences of this ubiquitous infection. Short-term protection of vulnerable patients may be achievable with normal immunoglobulin, but there are still considerable obstacles to the preparation and use of a human parvovirus vaccine.

A and B blood group antigen expression on mixed colony cells and erythroid precursors: relevance for human allogeneic bone marrow transplantation.

Using anti-A and anti-B blood group monoclonal antibodies and fluorescent activated cell sorting of human bone marrow, A (or B) blood group antigen was shown to be on 5.2 +/- 5.9 (mean +/- SD) % of CFU-GEMM and 12.5 +/- 19.6% of the erythroid burst forming cells (designated BFU-GEMM) as defined by the mixed colony assay, and 49.5 +/- 20% of the BFU-E and 83.5 +/- 9.9% of the CFU-E as defined by the erythroid colony assay. This antigen expression on the BFU-GEMM is consistent with the concept that erythroid bursts stimulated by leucocyte conditioned medium are less mature, and are closer in development to the pluripotent stem cell than the BFU-E. These results help to explain the delayed erythropoiesis, and perhaps impaired engraftment of all cell lineages, that may occur in some recipients of ABO incompatible bone marrow transplants with persistent and high anti-A titres.

Allogeneic bone marrow transplantation in a patient with acute myeloid leukemia secondary to Hodgkin's disease.

A patient with Hodgkin's disease entered complete clinical remission by combination radiochemotherapy. He developed dyshematopoiesis 1.5 years later and an overt acute nonlymphocytic leukemia 3 years after diagnosis. A complete remission was achieved following 2 courses of intensive polychemotherapy. Four months later, while still in remission, he underwent an allogeneic bone marrow transplantation (BMT) from an HLA-identical sister. Mild chronic graft versus host disease of the skin occurred 3 months after BMT, and now the patient has been in complete remission of leukemia for over 2 years. This appears to be a unique case of prolonged remission of a leukemia secondary to an intensively treated Hodgkin's disease.

Depletion of T lymphocytes in donor marrow prevents significant graft-versus-host disease in matched allogeneic leukaemic marrow transplant recipients.

For more than 15 years preclinical studies have suggested that acute graft-versus-host disease (aGvHD) might be prevented by the removal of immunocompetent T lymphocytes from the donor marrow inoculum. To test this observation in man 14 patients were given marrows virtually (greater than 99%) depleted of identifiable donor marrow T lymphocytes by the use of a "cocktail" of specific anti-T-cell monoclonal antibodies (MBG6 and RFT8) and rabbit complement. Patients were not given immunosuppressive prophylaxis after bone-marrow transplantation. Moderate to severe (grades II-IV) GvHD was totally prevented. 2 of 13 evaluable patients showed mild (grade I) skin GvHD only. Although peripheral blood recovery was slower than that obtained with other forms of GvHD prophylaxis, no fatal infections occurred. All patients survived the early post-transplant period.

The role of monoclonal antibodies in the prevention of graft versus host disease.

We have previously reported that the elimination of T-lymphocytes (greater than 99%) from the donor bone marrow prevents significant graft versus host disease (GvHD). This has been confirmed by other centres. Many of these groups have applied monoclonal antibodies with cytolytic rabbit complement. In some of these studies mostly patients with fully matched sibling donors have been studied and no further immunosuppression has been given during the regeneration period. The experience here shows that the haemopoietic regeneration (to recover a total leucocyte count of 1.0 X 10(9)/l) has only been minimally delayed (mean 25 days) when compared to patients receiving standard methotrexate (Mtx) GvHD prophylaxis (22 days). The incidence of cytomegalovirus (CMV) infections (14%; none fatal) was lower than that in our previous 54 patients (43% CMV infections, 13% fatal). Furthermore, no fatal pneumonitis was seen. The regeneration of T-cells in our patients has shown normal values of T8-positive cells in the circulation from 45 to 50 days onwards, as opposed to the previous groups of patients whose T8-positive cells regenerated to 3-4 times higher than normal values. These observations indicate that T-cell depletion with monoclonal antibodies is an effective method for preventing GvHD, but further studies are necessary to investigate the cause(s) of a new occasional complication, the rejection of the newly established bone marrow.